Process for producing L-proline by fermentation

ABSTRACT

A process for producing L-proline by fermentation, comprising culturing a microorganism belonging to the genus selected from Corynebacterium, Arthrobacter, Brevibacterium, Microbacterium and Saccharomyces and capable of producing L-proline in a culture medium to accumulate L-proline in the cultured liquor and recovering L-proline therefrom, which process is characterized by the use of a culture medium containing at least one member selected from L-glutamic acid and D-, L- and DL-pyrrolidonecarboxylic acid in association with a carbon source, nitrogen source and inorganic compounds.

This is a continuation of application Ser. No. 270,433 filed June 4,1981, now abandoned.

FIELD OF THE INVENTION

This invention relates to a process for producing L-proline by using amicroorganism. More particularly, this invention relates to a processfor producing L-proline by culturing a microorganism belonging to thegenus selected from Corynebacterium, Arthrobacter, Brevibacterium,Microbacterium and Saccharomyces and capable of producing L-proline in aculture medium to accumulate L-proline in the cultured liquor andrecovering L-proline therefrom.

DESCRIPTION OF PRIOR ART

Various processes for the production of L-proline by fermentation areknown. For example, L-proline is produced by culturing a wild strain, anutrient-requiring strain or a strain being resistant to a certainchemical agent selected from the bacteria of the genera Brevibacterium,Microbacterium, Micrococcus, Paracolobacterium, Bacillus, Escherichia,Kurthia, Microbacterium and Corynebacterium or a yeast of the genusSaccharomyces [Japanese Patent Publications 11751/68, 13679/68, 1193/69,1198/69, 6631/69, 26911/69, 38876/73, 28431/74, 33190/76, 40158/76,41386/79 and 105293/79]. Further, the process characterized by adjustingthe concentration of inorganic salts contained in the medium to aspecified range is disclosed in Japanese Patent Publication Nos.38557/71 and 42597/71. On the other hand, it is well known that thebiosynthesis of L-proline is effected via L-glutamic acid and that thesecretion of L-proline from a microorganism of Escherichia coli may beenhanced by addition of L-glutamic acid to the medium. However, thepractical value of such a process is very small because the amount ofthe secreted L-proline is very low (e.g. about 0.03 g/l) [Biochimica etBiophysica Acta, Vol. 104, 397 (1965)].

It has also been reported that the production yield of L-proline byfermentation of Kurthia catenaforma may be improved by addition of asurfactant and L-glutamic acid to the culture medium [AppliedMicrobiology, vol. 23, 758-764 (1972)]. However, it has never been knownthat an increased amount of L-proline may be accumulated in the culturedliquor when the fermentation is effected by using a group of certainbacteria (the socalled glutamic acid-producing bacteria such as thosebelonging to the genera Corynebacterium, Brevibacterium, Arthrobacter,and Microbacterium in a medium to which L-glutamic acid is added [J. ofthe Japanese Agricultural Chem. Soc., vol. 42, 703-710 (1968); AminoAcids and Nucleic Acids, No. 16, 126-133 (1967)].

SUMMARY OF THE INVENTION

This invention is based upon the discovery that it is possible toimprove the production yield of L-proline without addition of anysurfactant to the medium when a microorganism selected from the bactriaof the genera Corynebacterium, Brevibacterium, Arthrobacter, andMicrobacterium and a yeast of the genus Saccharomyces are incubated in aculture medium containing at least one member selected from L-glutamicacid and L-, D- and DL-pyrrolidonecarboxylic acid.

An object of this invention is to provide a process for producingL-proline by fermentation. L-proline is widely used for example for thepreparation of medicaments, foodstuffs and the like.

According to this invention, there is provided a process for producingL-proline by fermentation, comprising culturing a microorganism selectedfrom a bacterium and yeast, capable of producing L-proline, in a culturemedium to accumulate L-proline in the cultured liquor and recoveringL-proline from the cultured liquor, characterized by using a culturemedium which further contains at least one member selected fromL-glutamic acid, and D-, L- and DL-pyrrolidonecarboxylic acid, inassociation with a carbon source, a nitrogen source and inorganiccompounds.

The process of this invention will be fully and clearly described in thefollowing specification.

The microorganisms which may be used for the purpose of this inventioninclude L-proline producing microorganisms belonging to the generaCorynebacterium, Arthrobacter, Brevibacterium, Microbacterium andSaccharomyces, and the preferred strains are exemplified by

Corynebacterium glutamicum ATCC 21157

C. glutamicum ATCC 21158

C. glutamicum ATCC 21159

C. glutamicum ATCC 21355

C. acetophilum FERM-P 4045

C. acetoacidophilum FERM-P 4962

Arthrobacter citreus FERM-P 4963

Brevibacterium lactofermentum FERM-P 4964

Microbacterium ammoniaphilum FERM-P 4965

Saccharomyces cerevisiae ATCC 20169.

Either synthetic or organic medium may be used for the process of thisinvention, when the medium contains suitable amount of assimilablecarbon source, nitrogen source, inorganic substances and other nutrientsneeded for the growth of the microorganism used. Preferred carbonsources are exemplified by carbohydrates such as glucose, fructose,sucrose, sorbitol, glycerol, maltose, mannitol, starch hydrolyzate,molasses and the like; organic acids such as acetic acid, pyruvic acid,lactic acid, fumaric acid, citric acid and the like; and alcohols suchas e.g. methanol, ethanol etc. Preferred nitrogen sources areexemplified by ammonia; various inorganic and organic ammonium saltssuch as ammonium sulfate, ammonium chloride, ammonium phosphate,ammonium acetate and the like; various nitrogen-containing compoundssuch as urea and the like; peptone, meat extract, yeast extract, cornsteep liquor, casein hydrolyzate, soybean meal hydrolyzate, fermentedmicrobial bodies and hydrolyzate thereof. Examples of preferredinorganic substances include potassium dihydrogen phosphate, dipotassiumhydrogen phosphate, magnesium sulfate, sodium chloride, ferrous sulfate,manganese sulfate, calcium carbonate and the like. In addition,traceable amounts of nutrients such as biotin, thiamine, pantothenicacid and the like may, if desired, be used. In the case ofnutrient-requiring microorganism, it is necessary to add to the medium asuitable amount of such a nutrient required for the growth of themicroorganism. Although such addition may not be needed if the requirednutrient is inherently contained in the naturally-occuring substanceused as the nitrogen source.

The sources of L-glutamic acid and L-, D- and DL-pyrrolidonecarboxylicacid which may be added to the culture medium are exemplified byisolated L-glutamic acid and sodium glutamate and L-, D- andDL-pyrrolidonecarboxylic acid; L-glutamic acid fermentation liquor suchas glutamic acid fermentation broths, filtrate obtained by removal ofmicrobial cells from the fermentation broths, mother liquor obtained byseparating glutamic acid from the glutamic acid fermentation liquor,mother liquor obtained by separating sodium glutamate, beat molasses andwaste liquor thereof containing a large amount of glutamic acid orpyrrolidonecarboxylic acid as well as other substances containingglutamic acid or pyrrolidonecarboxylic acid.

The amount of glutamic acid or pyrrolidone carboxylic acid contained inthe medium may vary, depending upon the types of the used microorganismsand the time of addition of the acid and also upon the differingcomposition of the medium, and is usually more than 3 g/l of the medium.The upper limit may not be critical. However, at a concentration of morethan 150 g/l, negative effects of the salt used for adjusting the pH aswell as of the viscosity may be observed. The acid may be added to themedium at once or intermittently after the inoculation until themicroorganisms grow stationarily. The conditions for prolinefermentation may vary, depending upon the used microorganism, and thetemperature is preferably within a range of 25° to 40° C. During thefermentation, the pH of the medium is preferably kept at 6-9 for exampleby using inorganic or organic substance having acidic or alkaline pH,urea, calcium carbonate and the like. Under these conditions, thefermentation is effected aerobically so as to accumulate a largestpossible amount of L-proline, preferably for about 24-120 hours. Therecovery of L-proline from the fermented broths may be effected by theknown methods, such as the use of ion exchange resin, crystallizationfrom methanol and the like, in combination.

PREFERRED EMBODIMENTS

The following non-limiting examples illustrate the invention, whereinthe amount of proline was determined by the method reported in J. Biol.Chem., 199, 91-95 (1952).

EXAMPLE 1

A seed medium (30 ml) having the following composition was put in a 250ml Erlenmeyer flask, sterilized and inoculated with Corynebacteriumacetophilum FERM-P 4045. The fermentation was effected at 28° C. for 24hours with shaking (220 r.p.m.):

Meat extract: 10 g/l, Peptone 10 g/l, Yeast extract: 3 g/l, Sodiumchloride: 3 g/l (pH: 7.2).

The main medium had the following composition:

Waste molasses: 170 g/l (as glucose), Soybean meal hydrolyzate: 20 g/l,KH₂ PO₄ : 0.6 g/l, MgSO₄.7H₂ O: 0.6 g/l, (NH₄)₂ SO₄ : 23.4 g/l,FeSO₄.7H₂ O: 0.012 g/l, Thiamine hydrochloride: 100 μg/l, CaCO₃ : 30g/l, (pH=7.4).

To the main medium, a predetermined amount of sodium L-glutamate orDL-pyrrolidonecarboxylic acid as shown in Table 1 was added, and themedium (300 ml) was transferred to a 2 l Erlenmeyer flask equipped witha baffle on each occasion. After sterilization, each 30 ml of the saidseed culture was inoculated to each main medium which was then culturedat 28° C. for 4 days with shaking (220 r.p.m.). On each occasion, theaccumulated amount of L-proline is shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Accumulated amount of L-proline by addition of                                (A) sodium glutamate or (B) DL-pyrrolidone-                                   carboxylic acid (g/l of medium)                                               Added amount      A      B                                                    (g/l)             (g/l)  (g/l)                                                ______________________________________                                         0                25.1   24.8                                                  1                25.3   25.1                                                  3                26.9   27.7                                                 10                31.4   30.1                                                 20                35.6   34.8                                                 50                39.1   35.4                                                 90                40.5   36.6                                                 100               40.7   36.8                                                 ______________________________________                                    

EXAMPLE 2

Arthrobacter citreus FERM-P 4963, Microbacterium ammoniaphilum FERM-P4965, Brevibacterium lactofermentum FERM-P 4964, and Saccharomycescerevisiae ATCC 20169 were respectively treated by the method of Example1 except the use of a main medium having the following composition:

Glucose: 100 g/l, KH₂ PO₄ : 0.5 g/l, K₂ HPO₄ : 0.5 g/l, (NH₄)₂ SO₄ : 30g/l, Soybean meal hydrolyzate: 20 g/l, Biotin: 100 μg/l, MgSO₄.7H₂ O:0.25 g/l, FeSO₄.7H₂ O: 12 mg/l, MnSO₄.4H₂ O: 12 mg/l, ZnSO₄.7H₂ O: 10mg/l, Thiamine hydrochloride: 100 μg/l, CaCO₃ : 30 g/l, (pH=7.2).

Before use, the main medium was added with sodium L-glutamate (20 g/l)or L-pyrrolidonecarboxylic acid (20 g/l) to accumulate L-proline asshown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Accumulated amount of L-proline by addition                                   of (A) sodium glutamate or (B) L-pyrrolidone-                                 carboxylic acid (g/l)                                                         (1) Arthrobacter citreus FERM-P 4963                                          (2) Brevibacterium lactofermentum FERM-P 4964                                 (3) Microbacterium ammoniaphilum FERM-P 4965                                  (4) Saccharomyces cerevisiae ATCC 20169                                       Additive (g/l)     (1)    (2)     (3)  (4)                                    ______________________________________                                        nil      --        4.3    6.8     3.2  2.1                                    A        20        13.1   17.4    16.1 11.3                                   B        20        12.9   16.2    15.3 10.5                                   ______________________________________                                    

EXAMPLE 3

A similar treatment to that described in Example 1 was carried out byusing Corynebacterium glutamicum ATCC 21355 and a medium having thefollowing composition:

Glucose: 120 g/l, Urea: 3 g/l, Ammonium sulfate: 30 g/l, Ammoniumchloride: 30 g/l, KH₂ PO₄ : 1.5 g/l, K₂ HPO₄ : 0.5 g/l, MgSO₄.7H₂ O: 0.5g/l, FeSO₄.7H₂ O: 0.02 g/l, MnSO₄.4H₂ O: 0.02 g/l, Biotin: 50 μg/l,Thiamine hydrochloride: 1 mg/l, Peptone: 10 g/l, (pH=7.2).

24 hours after the beginning of the fermentation, paraaldehyde (40 g/l)was added to the medium. The fermentation was effected at 28° C. for 4days with shaking (220 r.p.m.) to accumulate 27.0 g/l of L-proline. Asimilar treatment effected with addition of 20 g/l of sodium glutamategave 38.9 g/l of the accumulated L-proline.

EXAMPLE 4

A seed medium (200 ml) having the following composition was put in a 2 lErlenmeyer flask equipped with a baffle and sterilized. After this,Corynebacterium acetophilum FERM-P 4045 or C. acetoacidophilum FERM-P4962 was inoculated to the medium and cultured at 28° C. for 24 hourswith shaking (220 r.p.m.).

Sucrose: 60 g/l, KH₂ PO₄ : 2 g/l, MgSO₄.7H₂ O: 0.5 g/l, FeSO₄.7H₂ O:0.01 g/l, MgSO₄.4H₂ O: 0.01 g/l, Corn steep liquor: 5 g/l, Thiaminehydrochloride: 100 μg/l, Biotin: 100 μg/l, Soybean meal hydrolyzate: 20g/l, Urea: 3 g/l, (pH=7.4).

To a main medium having the following composition was added a motherliquor obtained by crystallizing and separating sodium glutamate in anamount of 20 g/l calculated as glutamic acid. Each 700 ml of the mixturewas put in a 2 l jar fermentor and sterilized. After this, on eachoccasion, 200 ml of the seed culture was inoculated to the mixed mainmedium and cultured at 30° C. under aerobic condition. During thefermentation, the pH of the medium was adjusted to 7.0 by addition ofacetic acid solution automatically.

Sucrose: 20 g/l, Ammonium acetate: 7 g/l, MgSO₄.7H₂ O: 0.6 g/l, KH₂ PO₄:4 g/l, (NH₄)₂ SO₄ : 40 g/l, Soybean meal hydrolyzate: 20 g/l, FeSO₄.7H₂O: 0.012 g/l, MnSO₄.4H₂ O: 0.012 g/l, ZnSO₄.7H₂ O: 0.01 g/l, Biotin: 100μg/l, Thiamine hydrochloride: 100 μg/l, (pH=7.4).

72 hours after the beginning of fermentation, the consumed amount ofacetic acid was 15% on the basis of the initial volume of the medium andthe accumulated proline was 38.8 g/l by FERM P 4045 or 30.1 g/l byFERM-P 4962. For comparison, a similar treatment was carried out withoutaddition of the glutamic acid solution and the corresponding value ofproline was 25.4 g/l by FERM-P 4045 or 21.3 g/l by FERM-P 4962.

We claim:
 1. A process for producing L-proline by fermentation,comprising culturing a microorganism selected from the group consistingof Corynebacterium, Arthrobacter, Microbacterium and Saccharomyces in aculture medium containing at least one member selected from D-, L- andDL-pyrrolidonecarboxylic acid to accumulate L-proline in the culturedliquor, and recovering L-proline therefrom.
 2. The process of claim 1,in which the microorganism is selected from Corynebacterium glutamicum,Corynebacterium acetophilum, Corynebacterium acetoacidophilum,Arthrobacter citreus, Microbacterium ammoniaphilum and Saccharomycescervisiae.
 3. The process of claim 2, in which the microorganism isselected from the group consisting of Corynebacterium glutamicum ATCC21157, Corynebacterium glutamicum ATCC 21158, Corynebacterium glutamicumATCC 21159, Corynebacterium glutamicum ATCC 21355, Corynebacteriumacetophilum FERM-P 4045, Corynebacterium acetoacidophilum FERM-P 4962,Arthrobacter citreus FERM-P 4963, Microbacterium ammoniaphilum FERM-P4964 and Saccharomyces cerevisiae ATCC
 20169. 4. The process of claim 1,in which the culturing is effected at a temperature of from 25° to 40°C. and at a pH of from 6 to
 9. 5. The process of claim 1, in which atleast one member selected from the group consisting of D-, L- andDL-pyrrolidone-caroboxylic acid is present in an amount of from 3 to 150g/l of the medium.
 6. A process for producing L-proline by fermentation,comprising culturing a microorganism selected from the group consistingof Corynebacterium, Arthrobacter, Microbacterium and Saccharomyces in aculture medium containing glutamic acid without addition of a surfactantto accumulate L-proline in the cultured liquor, and recovering L-prolinetherefrom.
 7. The process of claim 6, in which the microorganism isselected from Corynebacterium glutamicum, Corynebacterium acetophilum,Corynebacterium acetoacidophilum, Arthrobacter citreus, Microbacteriumammoniaphilum and Sacchromyces cerevisiae.
 8. The process of claim 7, inwhich the microorganism is selected from Corynebacterium glutamicum ATCC21157, Corynebacterium glutamicum ATCC 21158, Corynebacterium glutamicumATCC 21159, Corynebacterium glutamicum ATCC 21355, Arthrobacter citreusFERM-P 4963, Microbacterium ammoniaphilum FERM-P 4965 and Saccharomycescerevisiae ATCC
 20169. 9. The process of claim 6, in which the amount ofglutamic acid is from 3 to 105 g/l of the medium.